Method for altering the binding specificity of plasma proteins by oxidation

ABSTRACT

The binding specificity of at least one plasma protein suspended or dissolved in a liquid medium is altered by exposing the protein to an oxidizing agent or an electric current sufficient to alter its binding specificity. A masked protein such as an autoantibody can be recovered from blood or blood products or extracts by oxidizing the protein to change its binding specificity.

This application is a continuation of U.S. application Ser. No.13/712,151, filed on Dec. 12, 2012, which is a continuation ofapplication Ser. No. 12/107,819 filed on Apr. 23, 2008, now U.S. Pat.No. 8,334,146, which is a continuation-in-part application of U.S.application Ser. No. 10/863,365, filed Jun. 9, 2004, now U.S. Pat. No.7,368,542, which claims the benefit of the filing date of U.S.Provisional Application No. 60/476,607, filed Jun. 9, 2003. U.S.application Ser. No. 12/107,819, now U.S. Pat. No. 8,334,146, is also acontinuation of application Ser. No. 11/359,489, filed Feb. 23, 2006,now U.S. Pat. No. 7,892,751, which is a continuation-in-part of U.S.patent application Ser. No. 11/108,826, filed on Apr. 19, 2005, whichapplication is a continuation-in-part of Ser. No. 10/863,365, filed Jun.9, 2004, now U.S. Pat. No. 7,368,542, which application claims thebenefit of U.S. Provisional Application Ser. No. 60/476,607, filed Jun.9, 2003, the contents of each of which are incorporated by referenceherein in their entireties.

FIELD OF INVENTION

The present invention relates to a method of altering a bindingspecificity of a plasma protein that has a binding specificity that canbe altered by oxidation-reduction reactions. The present inventionfurther relates to a method of obtaining autoantibodies by unmaskingautoantibodies naturally present in the blood, plasma or serum of normalsubjects.

BACKGROUND OF THE INVENTION

The term “autoimmune disease” refers to a group of diseases wherein theimmune system mistakenly attacks cells, tissues and organs of a person'sown body. Typically, autoimmune diseases involve antibody binding of thebody's own components, such as common proteins and lipids. Antibodiesthat bind to self-compounds (or, more typically, to compounds that areso common that they are found in every organism) are referred to asautoantibodies. As an example, autoantibody binding of phospholipidsand/or phospholipid-binding plasma proteins is associated with diseasessuch as systemic lupus erythematosus (SLE), deep vein and recurrentarterial thrombosis, pulmonary embolisms, recurrent spontaneousabortion, thrombocytopenia, chorea, epilepsy, livedo, idiopathicpulmonary hypertension, rheumatological conditions and a host ofcollagenous diseases. Other diseases associated with autoantibodiesinclude multiple sclerosis, Crohn's disease, discoid lupuserythematosus, Hashimoto's thyroiditis, psoriasis, diabetes andrheumatoid arthritis. There are about 80 different autoimmune diseases,and as a group, these diseases affect millions of people.

A conventional theory regarding the etiology of autoimmune diseases hasbeen that these diseases are caused by an overproduction ofautoantibodies in the diseased individual, possibly due to anoverexpression of a gene encoding such autoantibodies. According to thistheory, the blood of an affected individual contains an elevated levelof the particular autoantibody causing the disease, while the blood of anormal individual contains none of the autoantibody or only a trivialamount. This theory is seemingly supported by conventional assays, inwhich abundant autoantibodies can be detected in blood, or bloodproducts such as plasma or serum, from subjects having an autoimmunedisease, whereas only a zero or minimal amount of autoantibodies can bedetected in blood or blood products from subjects that do not have anautoimmune disease.

The present invention is based on the remarkable discovery, reportedherein, that blood from normal individuals in fact contains asignificant number of autoantibodies, in a wide variety of types andspecificities. It is possible to detect and isolate these autoantibodiesfrom blood or a blood product of a normal individual if the blood orblood product is treated by oxidation, by, for example with an oxidizingagent or electric current, according to a method described herein. Thisdiscovery of autoantibodies in significant quantities in normal blood ispreviously unreported and, to the best of the inventor's knowledge, theexistence of such autoantibodies in significant quantities in normalblood was completely unknown prior to the present invention.

Without being held to any particular theory, it is evident that ifautoantibodies may be obtained by manipulating normal blood taken frompersons who do not have any symptoms of autoimmune disease, then it mustbe that the immune system of normal persons routinely creates andcirculates these autoantibodies, but in some form wherein they aremasked or blocked, or otherwise prevented from having any harmfuleffects.

The discovery of autoantibodies in significant quantities in normalindividuals raises the question of why the autoantibodies are notdetected in a standard assay (typically based on the binding of anantibody to its corresponding antigen) and why the autoantibodies do notcause disease symptoms in normal individuals.

Based on earlier experiments described herein, a initial tentativeexplanation for how normal blood could contain autoantibodies withoutsuch antibodies being detected through ordinary screening procedures andwithout such antibodies causing disease, was that autoantibodies innormal individuals were somehow sequestered after they are produced. Forexample, the sequestration could be in the form of macromolecules suchas a low or high-density lipoproteins (LDL, HDL) or some other type ofmicroparticles, vesicles or micelles that could have the ability to keepautoantibodies cordoned off and separated from other components of thebloodstream. Under this theory, autoimmune disease could be triggered,not by the production of autoantibodies per se, but by the breakdown,disruption or lack of formation of the macromolecules, microparticles,vesicles or micelles sequestering the autoantibodies. This theory seemedsupported by the initial experiments wherein autoantibodies wereobtained from blood or serum samples after fairly drastic manipulationof the samples including shaking and heating.

In later experiments, described herein, however, it was shown thatsimpler methods of the invention, such as exposing blood or a bloodproduct to an oxidizing agent or to a DC electric current, can be enoughto obtain autoantibodies from normal blood, and that the process isreversible. Further, it was found that autoantibodies could be obtainedby treating commercial IvIg products, which would be free of any type ofmacromolecular sequestering entity. Based on these experiments, a morelikely theory for how normal blood could contain autoantibodies withoutsuch antibodies being detected through ordinary screening procedures andwithout such antibodies causing disease, is that the autoantibodiesfreely circulate along with other antibodies but that the antigenbinding site of autoantibodies is somehow blocked or inactivated innormal individuals. Under this theory, autoimmune disease could betriggered by oxidation to unblock the antigen-binding site ofautoantibodies. Further, this theory suggests a more general mechanismby which the binding specificity of certain plasma proteins may bealtered.

An immediate practical use of the discovery that forms the basis of thepresent invention is that it allows for an almost unlimited supply ofautoantibodies to be obtained, which autoantibodies can be used asstandards in diagnostic kits for the laboratory diagnosis of autoimmuneand other aPL-related diseases. Previously, collection of large amountsof autoantibodies for commercial use has been difficult because it wasthought that the autoantibodies had to be obtained from individualshaving an autoimmune disease or testing positive for autoantibodies instandard assays. The amount of such blood that can be obtained fromphlebotomy of individual patients or by pooling blood from a group ofpatients known to test positive for autoantibodies is limited. Othermethods of obtaining autoantibodies, such as screening phage librariesas described in U.S. Pat. No. 5,885,793, may be difficult andtime-consuming.

Testing blood samples for the presence or absence of masked antibodiesmay have important diagnostic value as it might presage or predict whatantibodies could appear subsequent to oxidative stress in particularindividuals

SUMMARY OF THE INVENTION

It is the object of this invention to provide a method of altering abinding specificity of a plasma protein that has a binding specificitythat can be altered by a change in its redox state.

It is a further object of the present invention to provide a method ofobtaining autoantibodies from blood, plasma or serum from normalindividuals.

It is a further object of the present invention to provide a method oftreating a subject having an autoimmune disease by administering to thesubject an antioxidant sufficient to inactivate autoantibodies in saidsubject.

It is a further object of the present invention to provide a method oftreating a subject having an autoimmune disease by inactivatingautoantibodies of said subject extracorporeally.

It is a further object of the present invention to provide a productcomprising a biological fluid or a protein-containing extract of abiological fluid that has been exposed to an oxidizing agent or a DCelectric current sufficient to alter the binding specificity of at leastone protein contained therein.

It is a further object of the present invention to provide a blood,plasma or serum sample from one or more persons who test negative forthe presence of autoantibodies in routine clinical assays and which hasbeen treated so that the blood, plasma or serum subsequently demonstratethe presence of autoantibodies.

These and other objectives are achieved by a method of altering abinding specificity of at least one circulating protein in a biologicalfluid or in a protein-containing extract of a biological fluid, thecirculating protein having a binding site with a binding specificitythat can be altered by a change in a redox state of the protein, byexposing the protein in the biological fluid or extract to an oxidizingagent or to a direct electric current (DC) to effect the alteration ofthe binding specificity of the circulating protein.

The objects are further achieved by a method comprising the steps ofproviding a composition comprising at least one plasma protein suspendedor dissolved in a liquid medium, the plasma protein having a bindingspecificity that can be altered by a change in its redox state, andexposing the composition to an oxidizing agent or a DC electricpotential sufficient to effect the alteration of the binding specificityof the plasma protein.

In another embodiment, the invention relates to a method of obtainingautoantibodies or other masked circulating proteins from a biologicalfluid or from an extract of a biological fluid by exposing theautoantibody or other masked circulating protein in the biological fluidor extract to an oxidizing agent or to a DC electric current sufficientto alter the binding specificity of the autoantibody or other maskedcirculating protein so that the autoantibody or other masked circulatingprotein becomes capable of binding to an antigen or ligand, therebybecoming detectable and recoverable from the biological fluid orextract, and recovering the autoantibody or other masked circulatingprotein from the biological fluid.

In another embodiment, the present invention relates to a method oftreating an autoimmune disease by administering to a subject having anautoimmune disease an amount of an antioxidant sufficient to inactivateautoantibodies in the subject. A treatment of a person having anautoimmune disease may include extracorporeal treatment of the blood toreduce the unmasked proteins and replace them as masked proteins.

In another embodiment, the present invention relates to a method ofscreening a normal individual's biological fluid or extract to determinewhich autoantibodies are masked and thus construct a potential antibodyprofile of autoantibodies that could cause autoimmune disease in thatindividual if exposed or unmasked by oxidation or an electromotiveforce.

As a particular, non-limiting example, blood, plasma or serum, or ablood extract such as an immunoglobulin mixture, may be exposed to anoxidizing agent or to a DC electric current to effect the alteration ofthe binding specificity of at least one autoantibody contained in theblood, plasma, serum or extract, so that the autoantibody becomesdetectable in and recoverable from the blood, plasma, serum or extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table listing the particular antiphospholipid antibodies(aPL) that were assayed by the in-house enzyme-linked immunosorbentassay (ELISA) format used in many of the Examples, described below.

FIG. 2 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples.

FIG. 3 is a composite table summarizing aPL assay results of bloodsamples from seven aPL-negative normal individuals, incubated accordingto the method described in the opening section of the Examples.

FIG. 4 is a table summarizing aPL assay results for a serum sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples, with the feature thathorse red blood cells (RBC) were substituted for human RBC in theprocedure.

FIG. 5 is a table summarizing aPL assay results of an incubation of aserum sample carried out according to a method described in the openingsection of the Examples, except that horse serum was substituted forhuman serum.

FIG. 6 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples, except that theincubation was carried out at room temperature (22° C.).

FIG. 7 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples, with the feature that0.7 mm Degalan (plastic) beads were used as the particulate solid in theincubation mixture.

FIG. 8 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples, except that theincubation mixture was kept stationary, instead of being shaken orrocked.

FIG. 9 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples, with the added featurethat the incubation mixture was heated to 56° C. for 30 minutes.

FIG. 10 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples, with the feature thata bacterial culture growth medium from a different supplier (BectonDickinson, Sparks, Md.) was used in place of the bacterial culturegrowth medium from Biomerieux.

FIG. 11 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples, with the feature thatthe incubation occurred under anaerobic conditions.

FIG. 12 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples, with the feature thatK562 cells (a human tumor cell line) were used instead of RBC.

FIG. 13 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, except that the bacteria culture growthmedium was replaced with a cell culture medium used for growing humancells.

FIG. 14 is a table summarizing aPL assay results for a cord blood samplefrom a normal aPL-negative mother and baby.

FIG. 15 is a table summarizing aPL assay results for a blood sample froma normal aPL-negative subject, incubated according to the methoddescribed in the opening section of the Examples, with the feature thatsodium nitroprusside (SNP) was used in place of RBC in the incubationmixture.

FIG. 16 is a table summarizing results of a lupus anticoagulant activityassay for a blood sample incubated according to the method described inthe opening section of the Examples. The blood sample was obtained froma subject whose blood is lupus anticoagulant negative prior toseroconversion by the present invention process.

FIG. 17 is a table listing other types of autoantibodies that have beenidentified in blood samples that are incubated according to the methoddescribed in the opening section of the Examples. The listed antibodieswere identified by immunofluorescence microscopy.

FIG. 18 is a graph showing the forward scatter (size) and side scatter(granularity) profile of the monocyte population of cells as defined fordensity gradient isolated human white blood cells by flow cytometry.

FIGS. 19 A-D are flow cytometry histograms showing monocyte activity ofvarious sera. In the histograms, antibody activity, if present, ismeasured by shifts in the median channel values (log scale) along thehorizontal axis. FIG. 19A shows monocyte reactivity of pooled normalhuman sera (NHS). FIG. 19B shows the monocyte activity of a serum from asingle normal subject. FIG. 19C shows the monocyte activity of a bloodsample from the subject shown in FIG. 19B that was treated according tothe method described in the opening section of the Examples. FIG. 19Dshows the monocyte activity of positive control sera.

FIG. 20 is a table summarizing the results of anti-nuclear-antibody(ANA) testing of various samples using a RELISA® screening assay.

FIG. 21 is a table summarizing aPL assay results for a sample of IvIgthat was incubated with hemin in a tris buffer.

FIG. 22 is a graph showing the amount of aPS, aCL, aPE, and aPC (asmeasured by optical density, OD) detected in a series of IvIgpreparations that were incubated with hemin, as a function of the amountof human serum (in μl) added to the preparations.

FIG. 23 is a graph showing the amount of aPS, aCL, aPE, and aPC (asmeasured by optical density, OD) detected in a series of diluted humanserum preparations that were incubated with hemin, as a function of theamount of hemin (in μl) added to the preparations.

FIG. 24 is a graph showing the amount of aPS, aCL, aPE, and aPC (asmeasured by multiples of the mean, MoMs) detected in a series of IvIgpreparations that were incubated with hemin and Vitamin C, as a functionof the amount of Vitamin C (in μg) added to the preparations.

FIG. 25 is a graph showing the amount of aPS, aCL, aPE, and aPC (asmeasured by multiples of the mean, MoMs) detected in a series of IvIgpreparations that were incubated with NaOH solubilized hemin,DMSO-solubilized hematoporphyrin IX (hpIX), NaOH-solubilized hpIX, NaOHalone, DMSO alone, and DMSO-solubilized hemin.

FIG. 26 is a graph showing the amount of aPS (as measured by opticaldensity, OD) detected in a series of IvIg preparations that wereincubated with increasing amounts of hemin and increasing amounts ofhemin and hemopexin (hpx).

FIG. 27 shows the Western blots obtained for three cell lysates withhemin-treated IvIg and untreated IvIg used as primary antibodies, alongwith a blot wherein anti-human HRP-tagged conjugate was used as acontrol.

FIGS. 28A and 28B are graphs showing the amount of aPL dependent and aPLindependent aPS, aCL, aPE, and aPC (as measured by multiples of themean, MoMs) detected in a series of IvIg preparations in whichelectrodes connected to a 9 volt battery were immersed in a phosphatebuffered saline solution containing the IvIg for 2 minutes.

FIG. 29 is a graph showing the amount of aPS, aCL, aPE, and aPC (asmeasured by multiples of the mean, MoMs) detected in a series of IvIgpreparations in which electrodes connected to a 6 volt battery wereimmersed in a phosphate buffered saline solution containing the IvIg for60 seconds.

FIG. 30 is a graph showing the amount of aPS, aCL, aPE, and aPC (asmeasured by multiples of the mean, MoMs) detected in a series of IvIgpreparations in which electrodes connected to a 6 volt battery wereimmersed in a phosphate buffered saline solution containing the IvIg, asa function of the time of immersion.

FIGS. 31A, 31B and 31C are graphs showing the amount of aCL, aPE, andaPS, respectively (as measured by multiples of the mean, MoMs), detectedin control solutions before and after exposure for 240 seconds toelectrodes connected to a 6 volt battery.

FIG. 32 is a graph showing the amount of aPS and aCL, respectively (asmeasured by multiples of the mean, MoMs), detected in the PBS-dilutedserum of an aPS and aCL-positive patient. In the experiment, graphiteelectrodes connected to a 6 volt battery were immersed in the dilutedserum for a variable amount of time.

FIG. 33 is a graph showing the amount of aPS, aCL, aPE, and aPC (asmeasured by multiples of the mean, MoMs), respectively, detected in thePBS-diluted serum of an aPE-positive patient. In the experiment,graphite electrodes connected to a 6-volt battery were immersed in thediluted serum for a variable amount of time.

FIG. 34 is a graph showing the amount of aPS, aCL and aPE, (as measuredby optical density, OD), respectively, detected in the PBS-diluted serumof an aPE-positive patient. In the experiment, the 10% adult bovineplasma (ABP) used in the determination of protein-dependent aPL bindingwas treated by immersing graphite electrodes connected to a 6-voltbattery in the ABP for a variable amount of time.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of altering the bindingspecificity of at least one plasma protein or circulating protein in abiological fluid or extract of a biological fluid.

As used herein, the terms “circulating protein” and “plasma protein” areused to refer to a protein naturally found in the circulation system ofanimals. Examples of circulating proteins include antibodies and otherplasma proteins. It is to be understood that the method of the inventionis not meant to apply universally to all plasma proteins or circulatingproteins, but rather applies to any plasma protein or circulatingprotein that has the property of having a binding specificity that canbe altered by a change in the redox state of the protein. The discoveryby the inventor that there are circulating proteins, such asautoantibodies, that have this property forms a basis of the invention.Examples of non-antibody proteins that have been found to have a bindingspecificity that can be altered by a change in the redox state includekininogen and prothrombin and/or beta2 glycoprotein.

The term “masked circulating protein” is newly coined for the presentinvention to designate and describe a circulating protein that, innormal individuals, is present in the blood, but is not detectable byconventional binding assays based on receptor-ligand binding because itsbinding site is, in the normal individual or in a sample taken from thenormal individual, masked or blocked or otherwise prevented from bindingan antigen, and that, when a sample containing the masked circulatingprotein is treated by changing its redox state, such as by exposure toan oxidizing agent or electric current according to a method of thepresent invention, becomes capable of binding an antigen and therebybecomes detectable in a sample. An example of a masked circulatingprotein is an autoantibody. As discovered by the present inventor,autoantibodies circulate in significant quantities in normal blood, butthey are not detectable in conventional assays based on antibody-antigenbinding. As discussed herein, an autoantibody becomes detectable andrecoverable when the autoantibody is subjected to oxidation-reductionconditions sufficient to alter its binding specificity. Autoantibodiesthat have been unmasked by oxidation include anti-phospholipid,anti-nucleolar (scleroderma associated), anti-lamins (very bright atnuclear pores), anti-mitochondrial (cytoplasmic), and anti-centrioleantibodies. Further, it has also been found that blood, serum or IvIgsamples that initially test negative for HCV (hepatitis C virus) testspositive for HCV after a treatment according to the present invention,suggesting that normal individuals have masked anti-HCV antibodies intheir circulation.

The term “altering the binding specificity” of a protein refers to aprocess whereby a protein is changed or altered, such as by oxidation orreduction, so that it becomes capable of specific binding of an antigenor ligand that it had not previously been capable of specificallybinding or becomes incapable of specific binding of an antigen or ligandthat it had previously been capable of specifically binding. The term“unmasking” refers to a process wherein the binding specificity of amasked circulating protein is altered so that the protein becomesdetectable by a binding assay based on the altered binding specificity.

The term “autoantibody” refers to any naturally occurring antibodyproduced by the immune system of an animal and that binds to aself-antigen, that is, to a compound or antigen produced by the animalitself.

The term “biological fluid” includes any bodily fluid that containscirculating proteins, including plasma, serum and whole blood, saliva,urine, lactation fluids and other secretions. The term“protein-containing extract of a biological fluid” refers to anypreparation that is collected or separated from a biological fluid, suchas immunoglobulin fractions. Blood, serum or plasma that may be used inthe present invention may be freshly obtained from an individual, or itmay be obtained from such sources as pooled blood or plasma preparationsobtained from blood banks or other blood collection facilities. For thepurposes of the present invention, the blood, serum or plasma may alsobe from collections that are out-of-date or otherwise found to besubstandard by blood banks or blood collection facilities. Although thisdescription focuses upon human blood, plasma and serum, the identicalprocess of this invention can be applied to animal blood and shouldresult in obtaining analogous animal antibodies for purposes relating toveterinary medicine. Preferably, blood or serum used in the method ofthe invention is diluted to reduce the effect of any antioxidants thatmay be contained in the blood, plasma or serum.

In the method of the present invention, the binding specificity of atleast one circulating protein or plasma protein in a biological fluid isaltered by exposing the protein to an oxidant or to an electric current.For example, the binding specificity of a masked circulating protein canby altered so that the protein is unmasked, that is, so that it is ableto bind an antigen that it was not able to bind before the method wascarried out. A protein that has had its binding specificity altered maythen be isolated and recovered by any separation method based onspecific binding.

If an oxidizing agent is used to carry out the method of the invention,the oxidizing agent can be any compound that is capable of altering theredox state of a biological molecule. More specifically, the oxidizingagent is a molecule that has the ability to be reduced by acting as anelectron acceptor for other molecules that act as electron donors.Examples of oxidizing agents include, but are not limited to hemin,chlorophyll, or other ring compounds containing a strong oxidizingmetal, and KMnO₄. Typically, when an oxidizing agent is used, a mixtureof the biological fluid or extract and the oxidizing agent must beincubated for a period of time, typically for about a day or overnight.The oxidizing agent should be used at a concentration sufficient enoughto alter the binding specificity of a protein having an alterablebinding specificity, but not at a concentration that might destroy theprotein. In the case of autoantibodies, it has been found that differenttypes of autoantibodies can interact differently with differentantioxidants. For example, for the unmasking of aPC autoantibodies, theresults are poor with hemin and very good with KMnO₄.

If a DC electric current is used to carry out the method of theinvention, the method may be carried out by any means of delivering anelectric current, such as by immersing positive and negative electrodesinto a conductive solution containing the sample to be treated.Typically, a solution containing a biological fluid may be exposed to anelectric potential of a sufficient magnitude and of a sufficientduration to alter the binding specificity of a protein having analterable binding specificity. It has been found that positive resultsmay be obtained by exposing a solution to an electric potential of 6-24volts for a few seconds to a few minutes. As discussed in the examples,an extended exposure to an electric current may result in reversibilityof the alteration of the binding specificity.

Attempts to produce positive results using an AC current have beenunsuccessful.

Without being bound to a specific theory, it is preferred, in the caseof an autoantibody, that the autoantibody be exposed to the oxidizingagent or electric current in an amount or for a time sufficient tooxidize an antigen binding site in a Fab portion of the autoantibody.

Whether a particular protein of interest is one that has a bindingspecificity that can be altered by changing its redox state and theeffectiveness of any set of conditions for altering the bindingspecificity of the particular protein of interest may be readilydetermined by ELISA or other ligand-receptor assays. Such assays can becarried out before and after a protein is subjected to redox conditionsto see whether the process has altered the binding specificity of theprotein. For example, the best oxidizing agent to recover a specificautoantibody can be readily determined by simple experimentation.

A further aspect of the present invention is the possibility of treatinga subject having an autoimmune disease, either by administering to thesubject an amount of an antioxidant sufficient to inactivateautoantibodies in the subject or by taking a blood sample from thesubject, exposing the blood sample to an antioxidant or electric currentsufficient to inactivate autoantibodies in said blood sample, andreturning the blood sample to the subject.

A further aspect of the present invention is a method of screening anormal individual's biological fluid or extract to determine whichautoantibodies are masked and thus construct a potential antibodyprofile of autoantibodies that could cause autoimmune disease in thatindividual if exposed or unmasked by oxidation or an electromotiveforce. For example, in general terms, a blood, plasma or serum samplefrom a subject can be assayed to determine an amount and/or type ofautoantibodies detectable in the sample. Thereafter, a blood, plasma orserum sample from the subject can be treated by exposing the sample toan oxidizing agent or a DC electric current, and the treated blood,plasma or serum sample from the subject can be assayed to determine anamount and/or type of autoantibodies detectable in the treated sample.Thereafter, the amount and/or type of autoantibodies detectable in thesample before the treating step can be compared with the amount and/ortype of autoantibodies detectable in the sample after the treating step.

It has been found that untreated blood, plasma, serum or IvIg samplesand blood, plasma or serum or IvIg samples treated according to themethod of the present invention can be lyophilized and shipped orstored. When the samples are reconstituted, they retain their respectiveactivity.

EXAMPLES

Having described the invention, the following examples are given toillustrate specific applications of the invention, including the bestmode now known to perform the invention. The examples are presented inapproximate chronological order and thus show a progression in theunderstanding of components and procedures required to achieve theeffects of the invention. These specific examples are not intended tolimit the scope of the invention described in this application.

Regarding each of the Examples 1-17 described herein, unless otherwisenoted, the following procedure was typically used: A 10 ml sample ofwhole blood or 5 ml of serum or plasma from a normal aPL-negativesubject and 4-5 ml of packed mammalian red blood cells were added to avial containing 30 ml of Biomerieux brand bacterial culture growthmedium (containing at least the following ingredients: distilled water,soybean-casein digest broth, yeast extract; dextrose; sucrose; hemin;menadione (vitamin K3); pyridoxal HCl (vitamin B6); and sodiumpolyanetholesulfonate (SPS) and charcoal. Then, the mixture wasincubated, with rocking or shaking, at 37° C. for a period of 18-22hours. Following the incubation and centrifugation, a sample of theincubated blood or serum/RBC was tested for the presence ofantiphospholipid antibodies (aPL) using a comprehensive in-house ELISAaPL format that provides 24 separate aPL test results. The testingprocedure is described in greater detail in the following publications,incorporated herein by reference: Wagenknecht D R, et al., TheEvolution, Evaluation and Interpretation of Antiphospholipid AntibodyAssays, Clinical Immunology Newsletter, Vol. 15, No. 2/3 (1995) pp.28-38 and McIntyre J A, et al., Frequency and Specificities ofAntiphospholipid Antibodies (aPL) in Volunteer Blood Donors,Immunobiology 207(1): 59-63, 2003.

FIG. 1 shows the 24 separate aPL specificities that were tested for byusing the comprehensive in-house ELISA aPL format. Four specificitieswere assessed, 1) aPS=antiphosphatidylserine, 2) aCL=anticardiolipin, 3)aPE=antiphosphatidylethanolamine, and 4) aPC=antiphosphatidylcholine.For each of these aPL specificities, three immunoglobulin isotypes weresought, IgG, IgA and IgM. Each specificity and each isotype wereassessed in the presence (dependent) and absence (independent) of abuffer diluent supplement, 10% adult bovine plasma (ABP), which containsthe phospholipid-binding plasma proteins) or 1% bovine serum albumin,(BSA, which is devoid of phospholipid-binding plasma proteins),respectively. The final dilution of the subjects' blood samples wasbetween 1/50 and 1/100.

The results in the 24 aPL specificities obtained for the variousexperiments described herein are given in the accompanying figures. Thepositive/negative findings are expressed in multiples of the means (MoM)based on testing plasma samples from 775 normal blood donors, asdescribed in McIntyre J A, Immunobiology, above. The presence of +++indicates strong antibody activity. The markers of + and ++ indicate lowand intermediate antibody activity, respectively. The figures alsoprovide the normal range values for each aPL specificity and isotypecombination.

A positive result in the column indicated as PL binding protein“dependent” means that the antiphospholipid antibody (aPL) is actuallybinding to a plasma protein that initially has bound to the particularphospholipid indicated. Plasma proteins that typically can be bound byPS and CL include the following: beta₂-glycoprotein I, prothrombin,protein C, protein S, annexin V, and complement components Factor H andC4 (see, for example, McIntyre, J. A., Wagenknecht, D. R. and Faulk, W.P. Antiphospholipid antibodies: Discovery, definition, detection anddisease. Prog. Lipid Res. 42(3): 176-237, at page 182). Thephysiological nature of the plasma protein binding is not knownprecisely for all of the phospholipids, but such binding is thought toinduce conformational changes in the plasma protein structure, therebyexposing novel or cryptic epitopes that then are targeted by theindividuals' autoantibodies. Plasma proteins that typically can be boundby phospholipid PE include the following: high and low molecular weightkininogens, and factor XI and prekallikrein. The latter two proteins canbe detected by virtue of their fidelity in binding to high molecularweight kininogen. The plasma proteins that bind to PC have not yet beendefined. In certain experiments, plasma-protein independent aPL areobserved (see FIG. 3). A possible explanation for this activity is thatit represents the presence of residual phospholipid-binding plasmaproteins that are present in the original blood sample.

Example 1

A sample of blood from a normal subject was incubated and testedaccording to the procedure described above. The results of the aPL ELISAare shown in FIG. 2. As shown in FIG. 2, the incubated blood sampleshows a dramatic presence of autoantibody activity, in comparison to thenormal, untreated blood shown in the Normal ranges column. Inparticular, strong autoantibody activity is shown in theprotein-dependent category for aPS (IgG), aCL (all isotypes), and aPE(IgG). The low or absent IgG aPC autoantibody activity was acharacteristic finding in the early examples and in procedures in whichhemin was used as the oxidizing agent. This result indicates thatautoantibodies to PC, especially of the IgG isotype are different andperhaps do not become activated in the same way as do the others. Inlater experiments, it was found that significant levels of aPC can bedetected in samples that were treated with KMnO₄ (data not shown).

Example 2

Blood samples drawn from seven healthy subjects were incubated andtested according to the procedure described above. In particular, allseven subjects' bloods were drawn within a 20 minute period andincubated for 20 hours in identical conditions. FIG. 3 is a compositetable showing the range of aPL seroconversion for these seven samples.These results show that there are variations in the aPL levels detectedas well as the isotypes present among different individuals.Nevertheless, as shown by the invention, each individual had aPLantibodies that could be detected after incubation.

Example 3

In a first experiment, a serum sample from a normal subject wasincubated and tested according to the basic procedure described above.In the incubation mixture, horse red blood cells (RBC) were used insteadof human RBC. The results of the aPL ELISA are shown in FIG. 4. As shownin FIG. 4, significant aPL activity was obtained, particularly withrespect to aPS (IgG and IgM) and aCL (IgA and IgM).

In a second experiment, a horse serum, instead of human serum, wasincubated with human RBC and tested according to the basic proceduredescribed above. The results of the aPL ELISA are shown in FIG. 5. Asshown in FIG. 5, aPL activity was not obtained. (The ELISA assay used inthis experiment utilized human-antibody-specific alkaline phosphataselabeled antibody probes to detect aPL, so whether the incubated samplecontained horse aPL is unknown.)

The results shown summarized in FIGS. 4 and 5 unequivocally demonstratethat all aPL that are obtained during the seroconversion process of thepresent invention originate from the human serum and are not releasedfrom the human RBC, since the first experiment uses horse RBC, which aredevoid of human antibodies, in place of human RBC and still showspositive results, whereas the second experiment uses horse serum in thepresence of human RBC and shows negative results.

Example 4

A blood sample from a normal subject was incubated and tested accordingto the basic procedure described above, except that the incubation wascarried out at room temperature (22° C.), instead of at an elevatedtemperature. FIG. 6 shows that the sample did not undergo seroconversionwhen incubated at room temperature. These results suggest that theprocess of seroconversion may be temperature sensitive.

Example 5

A blood sample from a normal subject was incubated and tested accordingto the basic procedure described above, with the feature that 0.7 mmDegalan (plastic) beads were used as the particulate solid in theincubation mixture instead of charcoal. Since charcoal was used ininitial experiments showing seroconversion, this experiment was carriedout to determine whether charcoal plays a specific role in theseroconversion. FIG. 7 shows that the sample exhibited seroconversioneven when plastic beads were used in place of charcoal. These resultssuggest that the role of charcoal is mechanical, rather than chemical,in nature, and that any particulate solid, such as plastic, resin orglass beads, can be used. Without being limited to any particulartheory, it can be theorized that the particulate component acts as anabrasive upon the RBC membrane, probably causing release of the NO ionfrom the RBC, either by interacting with the RBC AE1/Band 3 protein orwith the SNO-hemoglobin transition molecules or both. The possibility ofmechanical abrasion is supported by the observation in Example 6,wherein negative assay results are shown for an incubation mixture thatis not rocked or shaken. The particulate solids may also serve amechanical function of assisting autoantibody release.

Example 6

A blood sample from a normal subject was incubated and tested accordingto the basic procedure described above except that the incubationmixture was kept stationary, instead of being shaken or rocked. FIG. 8shows that the sample did not undergo seroconversion when it was keptstationary. These results suggest that movement may facilitateinteraction between solid particles and RBC. Stationary incubationconditions did not facilitate aPL release, although a small amount ofmovement such as produced by transport of the samples to the incubatormay produce small amounts of aPL release.

Example 7

A blood sample from a normal subject was incubated and tested accordingto the basic procedure described above, with the added feature thatafter incubation and removal of RBC and charcoal by centrifugation, theincubation mixture was heated to 56° C. for 30 minutes. FIG. 9 showsthat the amount of detected aPL was significantly increased by thisprocedure.

Example 8

A blood sample from a normal subject was incubated and tested accordingto the basic procedure described above, with the feature that abacterial culture growth medium from a different supplier (BectonDickinson, Sparks, Md.) was used in place of the bacterial culturegrowth medium from Biomerieux. FIG. 10 shows that the sample exhibitedseroconversion in the Becton Dickinson medium, indicating that themethod of the present invention is not dependent upon a bacterialculture growth medium from a particular source.

Example 9

A blood sample from a normal subject was incubated and tested accordingto the basic procedure described above, with the feature that theincubation occurred under anaerobic conditions (under nitrogen) insteadof under aerobic conditions (in the presence of oxygen and CO₂). FIG. 11shows that the sample exhibited seroconversion even under anaerobicconditions and that the method of the present invention is not dependentupon an aerobic environment.

Example 10

A blood sample from a normal subject was incubated and tested accordingto the basic procedure described above, with the feature that K562 cells(a human hematopoetic tumor cell line) were used instead of red bloodcells. Further, only 11.3 million K562 cells were present in the culturemedia, compared to 3-4 mls of packed RBC typically used in the method ofthe invention. FIG. 12 shows that the sample exhibited seroconversion.

Other experiments have shown that samples that are incubated with otherisolated cell types, lymphocytes, monocytes and neutrophils typically donot exhibit aPL seroconversion. In particular, white blood cells of thelymphoid and myeloid series did not support aPL release, nor did a cellline of porcine B lymphocytes designated as L14 (data not shown). Theseresults suggest that hemoglobin may be a key component in the incubationmixture, since K562 cells and RBC contain hemoglobin, and lymphocytes,monocytes and neutrophils do not.

Example 11

A blood sample from a normal subject was incubated and tested accordingto the basic procedure described above, except that the bacteria culturegrowth medium was replaced with a cell culture medium used for growinghuman cells: RPMI. FIG. 13 shows that seroconversion did not occur. Thisexperiment shows the importance of some ingredient in the bacterialculture media for the purpose of this invention. While RPMI is a culturemedia designed for human cells, it does not support aPL release whensubstituted for vial broth. Listings and comparisons of the ingredientsin the two different microbiology vial broths with RPMI show that RPMIlacks hemin and menadione (a man-made provitamin K) called vitamin K3.It is known that hemin is a porphyrin chelater of iron (Fe+++) derivedfrom RBC, and menadione is a fat-soluble vitamin. This indicates thatredox reactions may play a role in autoantibody release

Example 12

A placental cord blood sample incubated and tested according to thebasic procedure described above. The placental cord blood was drawnafter the birth of the baby, but before the placenta was detached fromthe uterine wall. Neither the mother's blood nor the baby's cord bloodshowed the presence of aPL in conventional laboratory assays. Whenprocessed according to the invention described herein, strong aPLantibody was demonstrated present in the cord blood samples, as shown inFIG. 14. The antibodies were IgG only, an observation that is compatiblewith antibodies of maternal origin. Since the mother transports IgG tothe fetus prior to birth, this experiment seems to indicate that themasked maternal autoantibodies transported to the fetus by way ofspecialized Fcγ receptors on the trophoblast (FcγRn) remain masked bythe fetus in the fetal blood. Since the mother's blood and the cordblood were shown to be aPL-negative prior to seroconversion by themethod of the invention, and since there were no IgM or IgAimmunoglobulins detected, these findings support the contention that theIgG aPL observed in the cord blood subsequent to seroconversion arematernal in origin. It also is of interest that the trophoblast thatexpresses the FcγRn does not express HLA antigens.

Example 13

A plasma sample from a normal subject was incubated and tested accordingto the basic procedure described above; with the feature that sodiumnitroprusside (SNP, 200 micromolar) was used in place of RBC in theincubation mixture. FIG. 15 shows that the sample exhibitedseroconversion.

Since SNP is a potent nitric oxide (NO) donor, these results providesupportive evidence that the NO radical is involved in the autoantibodyrelease and further support a theory that RBC and solid particulatesfulfill a role of providing NO⁻ donation from the RBC. Other freeradical mediated reactions apart from sodium nitroprusside may alsocause autoantibody release.

Example 14

A blood sample from a normal subject was incubated according to thebasic procedure described above and was tested for lupus anticoagulantactivity. Lupus anticoagulant or inhibitor is another type of aPL and istypically detectable only by functional laboratory assays. The resultsin FIG. 16 show a strong lupus anticoagulant (LA) in the seroconvertedblood taken from a lupus inhibitor negative individual and processed bythe method of this invention. While initially corrected by adding normalplasma to the seroconverted broth in the dRVVT assay, incubation for 1-2hours resulted in the reappearance of the inhibitor. This time frame isproposed as the time it takes for the LA or unmasked antibodies to bindthe relevant phospholipid-binding plasma proteins introduced by themixing study. It also rules out the possibility of clotting factordeficiencies since a 1:1 mix provides sufficient levels of clottingfactors to correct clotting times in a factor deficient sample. Thedilute prothrombin time (dPT) did not correct in the presence of normalplasma and increased prolongation of clotting times was observed afterincubation with normal plasma, which is indicative of a strong lupusinhibitor.

Example 15

Blood samples from five normal subjects were incubated according to thebasic procedure described above and were tested by fluorescencemicroscopy for the presence of other types of autoantibodies. Sera andplasma samples from these five individuals were negative prior toprocessing according to the teachings of the invention. FIG. 17 listsadditional autoantibody specificities identified by using the Hep-2 cellline. Identified were anti-nucleolar (scleroderma associated),anti-lamins (very bright at nuclear pores), anti-mitochondrial(cytoplasmic), and anti-centriole. The results show that autoantibodiesreleased by the method of the present invention can also be detected bya different methodology of detection, fluorescence microscopy, asopposed to ELISA-based testing. The results confirm that many types ofautoantibodies besides aPL are masked in the blood of individuals whoseserum and plasma test negative for these antibodies in routinelaboratory analyses.

From these results, it can be expected that many more autoantibodyspecificities await to be found by testing bloods processed by thisinvention.

Example 16

A blood sample from a normal subject was incubated according to thebasic procedure described above and was tested for reactivity withmonocytes using flow cytometry and fluorescent conjugated antihuman IgGantibodies. Comparative testing was done with untreated-pooled normalhuman sera (NHS), with serum from the same normal subject used with theinvention and with positive control human sera. (The treated bloodshowed no auto reactivity with lymphocytes and neutrophils; these dataare not shown.) FIG. 18 depicts the forward scatter (size) and sidescatter (granularity) profile of the normal subject's monocytepopulation of cells as defined by flow cytometry. This monocytepopulation of cells was confirmed by showing reactivity with CD 14monoclonal antibodies. FIG. 19A shows anti-monocyte reactivity with NHS.The median reactivity shown is 743.50 on a linear scale. FIG. 19B showsthe auto-anti-monocyte activity of the normal subject's serum; thissubject does not have antibody activity to autologous monocytes. Themedian reactivity shown is 737.00. FIG. 19C shows the auto-anti-monocyteactivity of a blood sample from the subject shown in FIG. 19B after itis treated according to the method of the invention. The median value isshown is 864.00, indicating strong auto-anti-monocyte activity. Despitethe fact that the plasma processed according to the teachings of theinvention were used at a dilution of 1/8, it showed more reactivity withmonocytes than did the undiluted positive control sera. Thus, thisexample shows that blood or serum samples processed according to themethod of this invention release autoantibodies that specifically targetmonocytes. The same results were documented for four additional samplesfrom other individuals when processed according to the teachings of theinvention.

Example 17

Comparative tests for the presence of anti-nuclear-antibodies (ANA)using a RELISA® screening assay were carried out on untreated cord bloodserum; cord blood incubated according to the method of the presentinvention, with no rocking; cord blood treated according to the methodof the present invention, with rocking; untreated serum from anANA-negative healthy donor (identified as ACS) and serum from the sameANA-negative healthy donor that was incubated according to the method ofthe present invention. As shown in FIG. 20, a significant amount of ANAwas identified in cord blood and serum samples that were treated by themethod of the present invention. From the results in FIGS. 16 and 17, itcan be expected that many more autoantibody specificities wait to befound by testing bloods processed by this invention.

Example 18

To understand the role of red blood cells in the phenomenon ofautoantibody release, experiments were designed to replace the red bloodcells with simpler ingredients that might mimic the action of the redblood cells. In the present experiment, the red blood cells and charcoalwere replaced with sodium nitroprusside (SNP) and ferric chloride. Thissubstitution was made because sodium nitroprusside is a powerful nitricoxide producer, and it is known that the RBC are carriers of NO⁻. Ferricchloride (FeCl₃ stock solution, 25 μM), was added as a substitute forthe iron in hemoglobin.

Culture bottles containing the bacterial culture growth medium and 5 mlof human plasma or serum and varying concentrations of sodiumnitroprusside (SNP, 200 μm) and exogenous ferric chloride (4.1 μm finalconcentration) was used in place of red blood cells and charcoal, wereincubated at 37° C. and then heated to 56° C. for 30 minutes. Thesamples showed seroconversion of aPL, but only IgG (data not shown).

The results suggest that NO⁻ may be involved in antibody unmasking, andsuggest that the mechanical action of a solid phase material in theculture bottle disrupts the red blood cells and releases NO⁻.Alternatively, the release or modification of NO may enable thehemoglobin molecule to participate in redox reactions.

Example 19

In an effort to determine whether the effect of unmasking autoantibodieswas due to the breakdown of autoantibody-containing macromolecularstructures within serum or blood or whether it was due to direct changesin the binding specificity of antibodies themselves, a series ofexperiments were carried out in which commercial intravenousimmunoglobulin (IvIg) was substituted for human plasma or serum.Commercial IvIg is an alcohol precipitate fraction of pooled plasma frommultiple donors, typically from 1,000-10,000 donors. Typically, IvIgcontains primarily IgG, and is mostly devoid of IgA, IgM and otherplasma proteins. When untreated IvIg is tested for the presence ofautoantibodies by ELISA testing, the test results are negative. Becauseof its manner of preparation, IvIg is also free of lipoprotein micelles,vesicles or other macromolecular structures. Therefore, if IvIg were totest positive for the presence of autoantibodies after an incubationtreatment, it would have to be that the autoantibodies were obtained byan alteration of IgG antibodies already present in the IvIg preparationand not by a breakdown of structures or vesicles concealing theautoantibodies.

In the examples that follow, the commercial preparation of IvIg used waslyophilized IvIg (Immune Globulin Intravenous (Human) Gammar-PI.V.,Aventis Behring, Kankakee, Ill.).

A 5 gram commercial preparation of lyophilized IvIg was reconstituted insterile phosphate buffered saline (PBS, 100 mg/ml). 1.7 ml of thereconsitituted IvIg solution was added to a culture bottle containingthe bacterial culture growth medium (without red blood cells orcharcoal) and was incubated at 37° C. for 20 hours. The incubatedmixture showed seroconversion and the presence of aPL IgG (data notshown). (As expected, only IgG was detected, not IgA or IgM.)

In similar experiments, autoantibodies were detected in a mixture thatwas incubated at room temperature in a shaking vial, but the resultswere not as good as at 37 degrees (results not shown).

Heating the IvIg-bacterial growth medium mixture above 37° C. did notresult in further increases in autoantibodies.

As a control, IvIg straight out of the bottle was tested for aPL andother autoantibodies, and the results were negative.

Example 20

In Example 19, it is shown that autoantibodies can be obtained byincubating a commercial IvIg preparation in a bacterial growth medium.The next step was to try to determine which ingredients in the bacterialculture growth medium play a role in producing detectableautoantibodies.

First, IvIg in 2% tryptic soy broth (TSB), (which contains peptones in a17 to 3 ratio of pancreatic digest of casein to papaya digest of soy,respectively) (the remainder being water) was incubated at 37° C. for 20hours with shaking. The incubated mixture was tested for the presence ofaPL, and the result was negative.

Next, IvIg was incubated in a test tube in soy broth, sodiumnitroprusside (SNP) and hemin (an iron (ferric) containingprotoporphyrin) at 37° C. for 20 hours with shaking. The amounts usedwere 60 microliters of IvIg, 5 microliters of SNP and 5 microliters ofhemin in a total of 1 ml of soy broth. The incubated mixture testedpositive for the presence of aPL, particularly aPS (15 MoM) and aPE (41MoM). (data not shown)

Example 21

A series of experiments was conducted to determine if incubation withhemin alone would be sufficient to cause the appearance ofautoantibodies in IvIg or in plasma or serum.

Reconstituted lyophilized IvIg (at a concentration of 100 mg/ml) wasadded to and incubated in a phosphate buffered saline (PBS) solutionwith hemin for 20 hours at 37° C. The amounts used were 300 μl of IvIgsolution and 5 μl of a hemin solution (75 μg) in a total volume 1 ml.

As shown in FIG. 21, the incubated mixture showed significant amounts ofaPS and aPE IgG, and, to a lesser extent, aCL IgG.

When serum or plasma were incubated with hemin under similar conditions,no autoantibodies were detected.

Example 22

The fact that positive results for the presence of autoantibodies couldbe obtained when IvIg was incubated with hemin, whereas negative resultswere obtained when serum or plasma were incubated with hemin suggestedthat serum or plasma could contain substances that inhibit or interferewith the process of obtaining autoantibodies.

In a series of experiments, IvIg was incubated in a Tris buffer withhemin, for 20 hours at 37° C., similar to the process of Example 21,with the added feature that an increasing amount of human serum (theinventor's) was added to the batches before incubation. Each separatebatch was tested for the presence of aPS, aCL, aPE and aPCautoantibodies, and the results are shown in FIG. 22. The results shownin FIG. 22 demonstrate that increasing amounts of serum did have aninhibitory effect on the release of antiphospholipid antibodies. Similarresults were shown with substituting plasma for serum (data not shown).A possible explanation for these results is that hemin, which containsan iron molecule in the ferric state and which is known as an activeoxidizing agent, may act to oxidize a binding site of certainimmunoglobulin molecules so that the altered binding site is able tobind self antigens. This process may be inhibited by substances, perhapsantioxidants, in the blood.

Example 23

Human serum (the inventor's) was diluted 1/10 in Tris buffer. In aseries of experiments, this diluted serum, in 1 ml batches, wasincubated with an increasing amounts of hemin, specifically, 0 μl, 10μl, 25 μl and 50 μl. (Previously, it had been found that hemin by itselfwas not sufficient to cause the release of autoantibodies from blood orserum, although it was sufficient to cause such release from IvIg.Therefore, the purpose of diluting the serum was to dilute the effect ofany interfering substances found in the blood, such as antioxidants.)The batches were tested for the presence of aPS, aCL, aPE and aPCautoantibodies, and the results are shown in FIG. 23. The results shownin FIG. 23 show that while no significant amounts of autoantibodies weredetected in diluted serum when 0 or 10 μl of hemin is added, significantamounts are detected with 25 μl of hemin. For an unknown reason, theamounts of detected autoantibodies were less with 50 μl of hemin.

Example 24

The next series of experiments was designed to determine if anantioxidant such as vitamin C, which is present in blood, would inhibitthe release of autoantibodies. In a series of experiments, IvIg wasincubated in a Tris buffer with hemin, with the added feature that anincreasing amount of ascorbic acid (Vitamin C) was added to thehemin-containing buffer and allowed to mix for 30 minutes before addingthe IvIg and before incubation. As shown in FIG. 24, there was about 78%inhibition of hemin-induced aPE release with 1 mg of Vitamin C, anamount that represents a physiological concentration of Vitamin C. Thereis a biphasic curve with aPS release, which raises the possibility thatVitamin C at low concentrations can act as an oxidizing agent, butbecomes an antioxidant (reducing) agent at higher concentrations.

Example 25

The next series of experiments was designed to determine whether thevehicle that hemin is dissolved in has an impact on the results obtainedand whether the iron atom in hemin is necessary. In a series ofexperiments, IvIg was incubated in a Tris buffer with hemin, or withother additives. In particular, in one instance, hemin was solubilizedwith NaOH. In another instance, it was solubilized with DMSO. In otherinstances, hematoporphyrin IX (hpIX), which is the same molecule ashemin, but without the iron (Fe+++), was used in place of hemin and wassolubilized with NaOH or DMSO. In other instances, NaOH and DMSO weretested as controls (without hemin or hpIX). As shown in FIG. 25, the useof NaOH solubilized hemin produced positive results for the presence ofautoantibodies, whereas hemin+DMSO, hpIX+NaOH, hpIX+DMSO, NaOH alone,and DMSO alone did not produce positive results.

Example 26

To further establish that hemin was causing oxidation of antibodies,equimolar amounts of hemopexin (Hpx) were added to the IvIg PBS heminmixture. Hpx is an antioxidant molecule with an extraordinarily highbinding affinity for heme iron. Lyophilized Hpx purchased from SciPac(Kent, England) was reconstituted in PBS at 10 mg/ml. Shown in FIG. 26are the aPS redox data resulting from adding increasing concentrationsof hemin to the IvIgG countered with adding equamolar concentrations ofHpx. Because there is an 1:1 binding interaction between hemin and Hpx,the Hpx was able to negate the redox capability of the ferric ironpresent in hemin.

Example 27

To illustrate the broad range and activity of autoantibodies that can beobtained by an oxidation treatment of IvIg, a series of Western blotswere set up using cell lysates from 3 different cell lines usinghemin-treated IvIg or untreated IvIg as primary antibodies and usinganti-human HRP-tagged conjugate as a control (HRP=horseradishperoxidase). The blots are shown in FIG. 27. The “B” lysate is a Blymphocyte cell line called Raja from a patient with a lymphoma. The “T”lysate is a T-lymphocyte-derived cell line called Jurkat again from aleukemic patient. The U87MG lysate is a glioblastoma blast cell line(brain cancer). The reduced lysates were run into the gel at 50 mg/mlconcentration. To obtain the hemin-treated IvIg preparation, 75 μg ofhemin was combined with 1 ml of PBS containing 6 mg of IvIgG. Incubationwas for 20 hours at 37 degrees. In FIG. 27, the blot whereinhemin-treated IvIg was used as the primary antibodies is labeled “TestIgG; the blot wherein untreated IvIg was used as the primary antibodiesis labeled “Control”, and the blot to which anti-human HRP-taggedconjugate was applied without primary antibodies is labeled “Secondary”.The hemin-treated and untreated IgG preparations were diluted 1/1000respectively. The anti-human HRP-tagged conjugate was used at a dilutionof 1/5000.

These data clearly show that the hemin-treated IvIg has abundantactivity towards human cellular components in comparison to untreatedIvIgG and the conjugate control, which do not.

Example 28

The next experiment was carried out to determine whether oxidizingagents other than hemin, and in particular, oxidizing agents that do notcontain iron, would be effective to unmask autoantibodies. A mixture of25 μg of potassium permanganate (KMnO₄) at a concentration of 100 μM,and 2 mg of IvIg in a total volume of 1 ml of phosphate buffered salinewas incubated overnight at 37° C. In the incubated mixture, aPC and aPScould be detected. aCL was usually detected, but not aPE (data notshown). It was later determined that a reason why aPE is not detected isbecause KMnO₄ alters the PE phospholipid antigen used in the ELISAtesting.

Example 29

After it was shown that autoantibodies could be unmasked by oxidationreactions, the next question was whether electrochemical methods, suchas an electromotive force from a battery, could achieve the same effect.

IvIg was dissolved in a phosphate buffered saline solution, and, inseparate experiments, galvanized steel, copper, or stainless steelelectrodes were connected to the positive and negative terminals of a9-volt battery and were submersed into the solution for 1-2 minutes.During this period, bubbling was noticed in the solution and the PBSsolution changed color (blue when copper wires were used, brown whenstainless steel wires were used and green when galvanized steel wireswere used). As shown in FIGS. 28A and 28B, the treated solution testedpositive for the presence of aPS, aCL, aPE and aPC autoantibodies, inaPL dependent testing, and positive for the presence of aPS, aPE and aPCautoantibodies in aPL independent testing.

Example 30

To avoid the interaction of metals with the solution and therebydetermine the effect only of an electric current, graphite electrodeswere used in place of the metal electrodes. Graphite is inert, but isable to pass electrons into conducting solutions without participatingin reactions.

IvIg was dissolved in a phosphate buffered saline solution, and graphiteelectrodes connected to the positive and negative terminals of a 6-voltbattery were submersed into the solution for 60 seconds. As shown inFIG. 29, the treated solution tested positive for the presence of aPS,aPE and aPC autoantibodies.

Example 31

In the experiments involving applying electric current to solutions ofIvIg in phosphate buffered saline, a significant increase in pH wasnoticed, possibly due to the formation of NaOH. In order to keep thereactions at physiological pH levels, a cell culture medium, RMPI, wassubstituted for the phosphate buffered saline.

The next series of experiments was carried out to determine the effectsof the time of exposure to the electric current on the unmasking ofautoantibodies. IvIg was dissolved in RMPI, a cell culture medium andgraphite electrodes connected to the positive and negative terminals ofa 6-volt battery were submersed into the solution for a variable amountof time. As shown in FIG. 30, the maximum release of dependent aPL wasobtained after 60 seconds of exposure to the current. Curiously, between2 minutes and 4 minutes, the amount of aPL declined or disappeared.

Example 32

Since the previous experiment had shown that aPL antibodies could beobtained from IvIg after exposure to an electric current, but that theaPL antibodies disappeared after further exposure to the current, thenext question was whether the unmasking of autoantibodies could bereversed by an electric current. That is, could a positive control serumbe treated so that autoantibodies were no longer detectable?

In separate experiments, aCL positive control serum, at a dilution of1:400, aPE positive control serum at a dilution of 1:75, and aPS at adilution of 1:400 were exposed to an electric current by immersinggraphite electrodes connected to the positive and negative terminals ofa 6-volt battery for up to 240 seconds. As shown in FIGS. 31A-31C, eachcontrol sera became negative for its respective specificity.

Example 33

Based on the results in Example 32, the next question that was asked waswhether autoantibodies of a patent having an autoimmune disease could beremasked if the patient serum was exposed to an electric current. Serumfrom a patient having elevated levels of aPS and aCL was diluted 1/400with phosphate-buffered saline (the dilution in PBS was in an amountthat would achieve an OD value of 1.000 in 10-15 minutes) and graphiteelectrodes connected to the positive and negative terminals of a 6-voltbattery were submersed into the solution for a variable amount of time.As shown in FIG. 32, the amount of aCL and aPS detectable in the samplesof the autoimmune patient's serum declined significantly after 30seconds and was no longer detectable after 2 minutes. These experimentswere repeated for other patients' antibodies and the same result wasobtained (data not shown).

Example 34

In an early experiment, a blood sample from a patient who had a veryspecific and high titer IgA aPE was exposed to hemin in a routinemicrobiology culture bottle. It was observed that after exposure tohemin her IgA aPE disappeared, and the emergence of IgG aPS, aCL andmost spectacularly, IgG aPE was detected in the aPL ELISA. At the time,an explanation for this phenomenon was not readily apparent.

With the discovery of a faster unmasking process using electric current,it became possible to confirm the earlier results with another patienthaving a high aPE. In this experiment, serum from a patient having ahigh aPE was diluted in PBS by 1/75 and graphite electrodes connected tothe positive and negative terminals of a 6-volt battery were submersedinto the solution for a variable amount of time. As shown in FIG. 33,the aPE became undetectable (masked) within 30 seconds of a 6-volt DCcurrent application, with a concomitant unmasking and detection of aPSand aCL IgG. The newly unmasked aPL peaked around 30 seconds only tobecome masked again after 2-4 minutes of exposure.

An important technical aspect addressed by the above experiment was thatthe patient aPE was treated apart from the plasma protein diluent usedin the assay, in the present case, 10% adult bovine plasma (ABP). Inother experiments not shown, the diluted patient sera were exposed to6-volt EMF conditions before adding the plasma proteins used in theELISA diluent. The important aspect of these experiments was to showthat the EMF effects were being applied to the patients' antibodies andnot to EMF changes in the plasma proteins used in the diluent.

These experimental data support the observations that redox reactionsare determining the appearance and disappearance of different antibodyspecificities. What is also learned from these experiments is that theredox effects appear to be limited to the antibody binding site(s), theFab portion of the antibody molecule. This is because the heterologousantihuman antibody-labeled conjugates used in the ELISA are not affectedas the conjugates continue to recognize the different antibody heavychain targets (Fc portions) of the antibody molecules. Thus, as thehuman antibody is not consumed or destroyed by redox, the most plausibleexplanation is that the antibody-binding site in the Fab portion of theantibody molecule contains accessible electrons that can participate inthe oxidation/reduction process.

Example 35

The next experiments were carried out to see whether plasma proteinsother than autoantibodies can have their binding specificity altered byoxidation-reduction. In these experiments, a 10% adult bovine plasma(ABP) solution, the same solution containing phospholipid-bindingproteins that had been used to determine protein-dependent aPL binding,was exposed to an electric current from a 6-volt battery for a variableperiod of time. The treated ABP samples were then used in ELISA assayswith aPS-, aCL- and aPE-positive patient sera to see if the treatment ofthe ABP would affect the outcome of the ELISA. As shown in FIG. 34, attime zero (untreated ABP), the positive patient sera give the aPLresponse in ABP that is routinely seen. As the 10% ABP is exposed tooxidation-reduction (EMF) over time, amount of aPL detected diminishesand after 2 minutes, the aPE positive serum is no longer positive. Theseresults indicate that the plasma proteins that are responsible for thepatients' aPL reactivity are altered by the exposure to the electriccurrent. For example, as kininogen is the plasma protein responsible forproviding a positive ELISA signal for aPE dependent reactions (thekininogen binds to PE, then the antibody binds to the kininogen, the aPEhowever will not bind to either PE or kininogen independently), thisshows that the kininogen in the ABP sample is being altered by the redoxexposure. aCL is also negative at 240 seconds exposure and since thispatient serum requires either prothrombin and/or beta2 glycoprotein (orboth could be involved) for producing a positive signal in the aPLELISA, these two proteins must also be altered by the redox reactions.The same two plasma proteins are involved in the aPS example.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

We claim:
 1. A method for screening an individual for autoimmunedisease, comprising the steps of: providing a composition comprising atleast one masked circulating protein suspended or dissolved in a liquidmedium, the at least one masked circulating protein having a bindingspecificity that can be altered by a change in its redox state, andexposing the composition to an oxidizing agent or DC current sufficientto effect the alteration of the binding specificity of the at least onemasked circulating protein.
 2. The method of claim 1 wherein said liquidmedium is diluted whole blood, serum, or plasma.
 3. The method of claim1 wherein the composition comprises intravenous immunoglobulin (IvIg)suspended or dissolved in a liquid medium.
 4. A diagnostic kit for thelaboratory diagnosis of autoimmune and other aPL-related diseasescomprising a standard including an antibody that has been oxidized byexposure to an oxidizing agent or DC current sufficient to effect thealteration of the binding specificity of the antibody.